Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway.
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway.
STAR Protoc. 2023 Dec 15;4(4):102662. doi: 10.1016/j.xpro.2023.102662. Epub 2023 Oct 27.
CRISPR-Cas9 gene editing is an efficient technique to modify specific sites/regions of DNA. Delivery of the Cas9 by mRNA is particularly promising in pre-clinical genome editing applications for its transient, nonintegrating feature. However, the off-target of Cas9-gRNA still remains a concern and needs a specific monitor. Here, we present a revised protocol to edit fibroblasts by in vitro transcribed Cas9 mRNA and profile its off-target effect by the optimized GUIDE-seq method. This protocol can also be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Ganna Reint et al. (2021)..
CRISPR-Cas9 基因编辑是一种高效的修饰特定 DNA 位点/区域的技术。信使 RNA （mRNA）递送 Cas9 因其瞬时、非整合的特性，在临床前基因组编辑应用中具有很大的应用前景。然而，Cas9-gRNA 的脱靶效应仍然是一个关注点，需要进行特定的监测。在这里，我们提出了一种用体外转录的 Cas9 mRNA 编辑成纤维细胞的改良方案，并通过优化的 GUIDE-seq 方法来分析其脱靶效应。该方案也可应用于其他细胞系。如需了解该方案的详细使用和执行方法，请参考 Ganna Reint 等人（2021）的研究。
